This K22 Transition Career Development Award is designed to provide support to help facilitate career and research development. My (Brian Barth, Ph.D.) training thus far in the fields of lipid biochemistry, normal and malignant cell biology, and nanomedicine, has prepared me for a career as an independent academic researcher. My career goals are to obtain additional training to prepare me for a career as an independent tenure-track scientist, and to establish a dynamic research program at an academic institution that focuses on leukemia biology and experimental therapeutics. My research goals are to identify sphingolipid- and redox- mediated mechanisms that can be exploited for the development of translational anti-leukemia therapeutic strategies. This K22 proposal will provide me the necessary resources to accomplish these goals, which will make me more competitive for future R01 funding. This proposal provides a career training plan that includes informal mentoring from an advisory committee, didactic training, teaching and mentoring of others, as well as defined milestones. My advisory committee is a strength of the application, consisting of noted experts that will ensure the fulfillment of my career development plan. Of particular note, this plan will include research laboratory training in each of their respective laboratories. This K22 proposal also includes a novel research project focused on evaluating alterations of ceramide metabolism during the progression of acute myeloid leukemia (AML), as well as a study of activation of the NADPH oxidase (NOX) as a mechanism mediating resistance to ceramide-based therapeutics. The most common type of leukemia in adults is AML, and an understanding of the underlying molecular mechanisms that mediate its underlying biology is essential to the development of better therapeutic strategies. Ceramide is a bioactive sphingolipid long implicated as a regulator of cellular stress and apoptosis. However, recent evidence has emerged challenging this dogma and suggesting that ceramide species are elevated in primary malignant specimens and as a function of disease stage/prognosis. NOX enzymes have gained notoriety as key players in normal cellular homeostasis as well as a variety of pathological processes. NOX enzymes play key roles in the regulation of cellular processes by modulation of redox-sensitive phosphatases and transcription factors, and have been shown to be stimulated by ceramide. NOX has also been shown to affect proliferation in AML, influence monocytic AML subtypes ability to interfere with normal immunity, and to affect oxidative signaling in AML. In this research study, upregulation of ceramide metabolism during the progression of AML will be investigated. Preliminary data indicates that flux through the ceramide metabolic pathway is substantially enhanced in AML as compared with the pre-leukemic condition myelodysplastic syndrome (MDS). The specific aims of this research study are to: 1) evaluate elevated ceramide metabolism during the progression of AML, and 2) evaluate NOX activation as a pro-leukemogenic pathway mediated by ceramide in AML. It is hypothesized that ceramide metabolism will be elevated as AML develops and progresses, such as the transformation of MDS to AML or the relapse of AML itself. This upregulation of ceramide metabolism would contribute mitogenic and survival advantage. It is further hypothesized that elevation in ceramide will trigger NOX activity that will augment pro-growth and pro- survival signaling pathways otherwise upregulated by leukemogenic transformations. To test these hypotheses, ceramide metabolism and sensitivity to nanoliposomal ceramide will be evaluated in patient materials representing the spectrum of the developing and progressing disease, and animal models engrafted with human AML will be used to study the evolution of the disease. More so, pharmacological or molecular inhibition of NOX will be used to reprogram ceramide-mediated apoptosis of AML. Overall this K22 proposal will provide for: 1) career development plan that with allow me to establish myself as an independent translational leukemia researcher, and 2) research support that will demonstrate a role for elevated ceramide metabolism in the development and progression of AML, as well as a role for ceramide-activated NOX in the pathobiology of AML. Finally, this proposal will lead to the development of NOX inhibitors as agents to combine with ceramide-based anti-AML therapeutics.